Selenium-rich lactobacillus rhamnosus microecological preparation as well as preparation method and application thereof

Abstract

The invention discloses a selenium-enriched lactobacillus rhamnosus microecological preparation as well as a preparation method and application thereof. The preparation method comprises the following steps: 1, inoculating lactobacillus rhamnosus SHA113 into a liquid culture medium to obtain a bacterial suspension; 2, adding sodium selenite into the bacterial suspension in the step 1, and reacting to obtain selenium-enriched lactobacillus rhamnosus; 3, centrifuging the selenium-rich lactobacillus rhamnosus obtained in the step 2, discarding the supernatant, and collecting the precipitate; 4, washing the collected precipitate with sterile water; 5, carrying out crushing treatment on the washed precipitate; and 6, drying the crushed precipitate into powder to obtain the selenium-rich microecological preparation, the selenium content in the obtained selenium-rich microecological preparation is 10mg / g to 35mg / g, and the selenium-rich microecological preparation is used for preparing medicines and health-care products for relieving liver injury in a low-pressure and low-oxygen environment and medicines and health-care products for relieving altitude stress and improving the exercise ability of an organism in a plateau hypoxia environment.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a selenium-enriched Lactobacillus rhamnosus microecological preparation and a preparation method and application thereof. Background Art

[0002] The plateau hypoxic environment has a significant impact on the physiological functions of the human body, and can easily cause problems such as fatigue and decreased athletic ability. People who work in plateau environments for a long time, such as soldiers, athletes, and outdoor workers, face serious health challenges. In order to cope with these problems, it is particularly important to develop effective anti-fatigue methods. Existing studies have shown that oxidative stress is one of the important causes of fatigue in the plateau hypoxic environment. Oxidative stress can cause lipid peroxidation of cell membranes, damage cell function, and then cause fatigue in the body. Therefore, substances with antioxidant effects have important application value in alleviating fatigue caused by the plateau hypoxic environment.

[0003] Selenium is an important trace element with significant antioxidant properties. In the body, selenium plays an antioxidant role by participating in the activity of enzymes such as glutathione peroxidase, protecting cells from oxidative damage. However, the low bioavailability of traditional selenium preparations in the body limits their application effect. In recent years, biological selenium has become a hot topic in antioxidant research due to its high bioavailability and excellent antioxidant properties.

[0004] Lactobacillus rhamnosus is a common probiotic that is widely present in the human intestine and has good biocompatibility and safety.

[0005] A low-pressure and low-oxygen environment can lead to abnormalities in indicators such as catalase, glutathione, and superoxide dismutase in the liver, causing damage to the organism. The existing technology lacks microecological preparations with high bioavailability and excellent antioxidant properties to alleviate this damage. Summary of the invention

[0006] The purpose of the present invention is to prepare a selenium-rich microecological preparation by a biosynthetic method to solve the problems existing in the prior art such as damage to the liver of an organism caused by a low-pressure and low-oxygen environment and decreased body movement ability caused by hypoxia.

[0007] The technical solution adopted by the present invention is:

[0008] A method for preparing a selenium-enriched Lactobacillus rhamnosus microecological preparation comprises the following steps:

[0009] Step 1: inoculating Lactobacillus rhamnosus SHA113 into a liquid culture medium to obtain a bacterial suspension;

[0010] Step 2: adding sodium selenite to the bacterial suspension in step 1 to obtain selenium-enriched Lactobacillus rhamnosus;

[0011] Step 3: centrifuge the selenium-enriched Lactobacillus rhamnosus obtained in step 2, discard the supernatant, and collect the precipitate;

[0012] Step 4: Wash the collected precipitate with sterile water;

[0013] Step 5: crushing the washed precipitate;

[0014] Step 6: Dry the crushed precipitate into powder to obtain a selenium-rich microecological preparation.

[0015] Furthermore, the Lactobacillus rhamnosus SHA113 in the step 1 was deposited in the China Center for Type Microorganism Collection on December 25, 2017, with a deposit number of CCTCC NO: M 2017838.

[0016] Furthermore, in the step 1, the inoculation amount of Lactobacillus rhamnosus is 1% v / v to 10% v / v, and the culture conditions are static culture at 37° C. or culture at 50 rpm to 250 rpm for 24 to 48 hours.

[0017] Furthermore, in the step 2, the concentration of sodium selenite is 1.0 mM to 10.0 mM.

[0018] Furthermore, in step three, the centrifugal speed is 4000 rpm / min to 10000 rpm / min, and the centrifugal time is 3 min to 10 min.

[0019] Furthermore, in the step five, the crushing method is ultrasonic crushing or grinding crushing.

[0020] Furthermore, the selenium content in the selenium-rich microecological preparation obtained in step six is ​​10 mg / g to 35 mg / g.

[0021] A selenium-enriched Lactobacillus rhamnosus microecological preparation prepared by any of the aforementioned preparation methods.

[0022] The selenium-enriched Lactobacillus rhamnosus microecological preparation is used in the preparation of drugs and health products for alleviating liver damage in a low-pressure and low-oxygen environment.

[0023] The selenium-enriched Lactobacillus rhamnosus microecological preparation is used in the preparation of medicines and health products for alleviating altitude sickness and improving the body's motor ability under high altitude hypoxia.

[0024] The beneficial technical effects of the present invention are as follows:

[0025] 1. The present invention provides a selenium-rich microecological preparation synthesized by Lactobacillus rhamnosus SHA113. The selenium-rich microecological preparation prepared by fermentation, separation and ultrasonic treatment processes has the characteristics of uniform particle size and good dispersibility. Animal experiments show that taking the selenium-rich microecological preparation prepared by the present invention for 4 consecutive days in advance can significantly enhance the athletic endurance of animals in plateau environments and reduce fatigue. High antioxidant properties, selenium-rich microecological preparations have good antioxidant properties, can effectively inhibit lipid peroxidation, and reduce oxidative stress damage to cells. Improve plateau physiological state, selenium-rich microecological preparations can effectively improve the body's physiological state in a plateau hypoxic environment and enhance anti-fatigue ability.

[0026] 2. The present invention has a good application prospect in the development of health foods, medicines, special medical foods, and dietary nutritional supplements that can alleviate liver damage in low-pressure and low-oxygen environments, alleviate altitude sickness, and improve the body's motor ability in high-altitude hypoxic environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a solid selenium-rich microecological preparation powder.

[0028] Figure 2 This is a comparison chart of the exercise endurance time of mice on the rotating rod.

[0029] Figure 3 A comparison of superoxide dismutase in mouse liver.

[0030] Figure 4 Comparison of glutathione peroxidase in mouse liver.

[0031] Figure 5 A comparison chart of malondialdehyde in mouse liver.

[0032] Figure 6 This is a comparison chart of lactic acid content in mouse serum.

[0033] Figure 7 This is a comparison chart of urea nitrogen content in mouse serum.

[0034] Figure 8 This is a comparison chart of creatine kinase content in mouse serum.

[0035] Fig. 9 This is a comparison chart of mouse liver glycogen content.

[0036] Fig.10 This is a comparison chart of mouse muscle glycogen content. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] The present invention discloses a selenium-enriched Lactobacillus rhamnosus microecological preparation and its preparation method and application. The selenium content in the preparation is 35 mg / g, which is obtained by adding sodium selenite to Lactobacillus rhamnosus SHA113 culture medium, fermentation, centrifugal collection, cleaning, crushing and drying. According to the dosage of 0.12 mg / kg per day, mice are gavaged with the preparation, once a day, for 4 consecutive days, and the exhaustion running time of mice under a simulated plateau hypoxia environment (4000 meters above sea level) can be extended by 4 times, while significantly reducing the lactic acid content, urea nitrogen content, and creatine kinase level in the blood, and significantly improving muscle glycogen and liver glycogen levels, meeting the requirements of anti-fatigue food. The preparation can be used alone, or it can be used in combination with milk powder, plant extracts, vitamins, dietary fiber, protein powder, etc., and has a good application prospect in the preparation of health foods, medicines, special medical foods, and dietary nutritional supplements that relieve altitude sickness and improve the body's motor ability under plateau hypoxia.

[0039] Embodiment 1:

[0040] A method for preparing a selenium-rich microecological preparation using Lactobacillus rhamnosus SHA113 comprises the following steps:

[0041] Step 1: Lactobacillus rhamnosus SHA113 was inoculated into MRS broth medium at an inoculation amount of 1% v / v, and cultured at 37° C. for 48 hours to obtain a fermented bacterial suspension.

[0042] Step 2: Add 3.0 mM sodium selenite to the fermented bacterial suspension in step 1, and continue to culture at 37° C. for 24 hours to obtain brick-red selenium-enriched Lactobacillus rhamnosus.

[0043] Step 3: Centrifuge the brick-red selenium-enriched Lactobacillus rhamnosus obtained in step 2 at 8000 r / min for 5 minutes in a centrifuge, discard the supernatant, and collect the brick-red bacterial sludge precipitate.

[0044] Step 4: Wash the collected brick-red bacterial sludge precipitate three times with sterile water of equal fermentation volume.

[0045] Step 5: Resuspend the washed red bacterial mud sediment in sterile water with a volume of 1 / 10 of the original bacterial suspension fermentation liquid, and place it in an ice bath for ultrasonic treatment. The ultrasonic parameters are: ultrasonic for 2 seconds, interval of 2 seconds, total ultrasonic time of 60 minutes, ultrasonic power of 500W, to obtain a selenium-rich microecological preparation suspension.

[0046] Step 6: Pre-freeze the selenium suspension of the selenium-rich microecological preparation obtained in step 5 in a -80°C refrigerator, and place it in a vacuum freeze dryer for drying after it is completely solidified. After all free water is removed, a selenium-rich microecological preparation powder with a selenium concentration of 35 mg / g is obtained.

[0047] Solid selenium-rich microecological preparation powder Figure 1 As shown. It shows the characteristics of uniform particle size and good dispersion.

[0048] Example 2 Efficacy Verification:

[0049] Preparation of selenium-rich microecological preparation suspension: Selenium-rich microecological preparation powder with a concentration of 35 mg / g and sterile water were mixed into a selenium-rich microecological preparation suspension with a concentration of 0.012 g / L for subsequent animal experiments.

[0050] Animal grouping. First, the animals were divided into three groups according to their body weight, namely the control group, the low-pressure and low-oxygen group, and the selenium-treated group. The control group was gavaged with sterile water, and an exhaustive running experiment was conducted in a normal pressure and normoxic environment. The selenium-treated group was gavaged with a selenium-rich microecological preparation suspension at a concentration of 0.012g / L, and the gavage dose was 0.12mg / kgBW, and an exhaustive running experiment was conducted in a low-pressure and low-oxygen environment. Three training sessions were conducted the day before the rotating rod exhaustive running experiment, with a speed of 20rpm / min. After training, mice that learned to run were selected to conduct a rotating rod exhaustive running experiment in a high-altitude hypoxic environment.

[0051] Rotarod exhaustion test for mice. One hour after the last oral administration, the mice were placed in a hypobaric and hypoxic chamber simulating an altitude of 4,000 meters to conduct a rotarod fatigue endurance test. The rotation speed of the rotarod was 30 rpm / min, and the exercise endurance time of each mouse on the rotarod was recorded as the mouse's anti-exercise fatigue endurance time. The results are shown in the figure below. Figure 2 As shown, compared with the mice in the control group, the exhaustion running time of the mice in the hypobaric hypoxia group in the plateau hypoxic environment was significantly reduced** (P=0.0053, N≥6). Compared with the mice in the hypobaric hypoxia group, the exhaustion running time of the mice in the selenium treatment group in the plateau hypoxic environment was significantly increased* (P=0.0393, N≥6).

[0052] Sampling. After resting for 60 minutes after exercise, blood was collected from the eyeballs of all mice. After standing at room temperature for 4 hours, the blood was centrifuged at 3000r / min for 15 minutes, and the upper serum was taken for subsequent testing. Fresh liver and skeletal muscle samples were rinsed with physiological saline and dried with filter paper for subsequent testing.

[0053] Determination of superoxide dismutase activity in the liver. The activity of superoxide dismutase in the liver of mice was tested using a kit, and the results were as follows: Figure 3As shown, compared with the mice in the control group, the superoxide dismutase activity of the mice in the hypobaric hypoxia group after exhaustive running in the high altitude hypoxia environment was significantly reduced* (P=0.0311, N≥6). Compared with the mice in the hypobaric hypoxia group, the superoxide dismutase activity of the mice in the selenium treatment group after exhaustive running in the high altitude hypoxia environment was significantly increased* (P=0.0262, N≥6).

[0054] Determination of glutathione peroxidase activity in liver. The activity of glutathione peroxidase in mouse liver was tested using a kit. The results are shown in the following table. Figure 4 As shown, compared with the mice in the control group, the glutathione peroxidase activity of the mice in the hypobaric hypoxia group after exhaustive running in the high altitude hypoxia environment was significantly reduced**(P=0.0030, N≥6). Compared with the mice in the hypobaric hypoxia group, the glutathione peroxidase activity of the mice in the selenium treatment group after exhaustive running in the high altitude hypoxia environment was significantly increased*(P=0.0276, N≥6).

[0055] Determination of malondialdehyde content in liver. The malondialdehyde content in mouse liver was tested using a kit, and the results were as follows: Figure 5 As shown, compared with the mice in the control group, the malondialdehyde content of the mice in the hypobaric hypoxia group after exhaustive running in the high altitude hypoxia environment was significantly increased* (P=0.0194, N≥6). Compared with the mice in the hypobaric hypoxia group, the malondialdehyde content of the mice in the selenium treatment group after exhaustive running in the high altitude hypoxia environment was significantly reduced* (P=0.0130, N≥6).

[0056] Determination of lactic acid content in serum. The lactic acid content in mouse serum was tested using a kit, and the results were as follows: Figure 6 As shown, compared with the mice in the control group, the lactic acid content of the mice in the hypobaric and hypoxic group after exhaustive running in the high altitude hypoxic environment was significantly increased* (P=0.0369, N≥6), and the lactic acid content of the mice in the selenium treatment group after exhaustive running in the high altitude hypoxic environment was significantly reduced**** (P<0.0001, N≥6). Compared with the mice in the hypobaric and hypoxic group, the lactic acid content of the mice in the selenium treatment group after exhaustive running in the high altitude hypoxic environment was significantly reduced**** (P=0.0002, N≥6).

[0057] Determination of urea nitrogen content in serum. The urea nitrogen content in mouse serum was tested using a kit, and the results were as follows: Figure 7 Compared with the mice in the control group, the urea nitrogen content of the mice in the hypobaric and hypoxic group after exhaustive running in the high altitude hypoxic environment was significantly increased** (P=0.0083, N≥6). Compared with the mice in the hypobaric and hypoxic group, the urea nitrogen content of the mice in the selenium treatment group after exhaustive running in the high altitude hypoxic environment was significantly reduced* (P=0.0213, N≥6).

[0058] Determination of creatine kinase content in serum. The kit was used to test the creatine kinase content in mouse serum. The results were as follows: Figure 8 As shown. Compared with the mice in the control group, the creatine kinase content of the mice in the hypobaric and hypoxic group after exhaustive running in the high altitude hypoxic environment was significantly increased****(P<0.0001, N≥6). Compared with the mice in the hypobaric and hypoxic group, the creatine kinase content of the mice in the selenium treatment group after exhaustive running in the high altitude hypoxic environment was significantly decreased***(P=0.0123, N≥6).

[0059] Determination of liver glycogen content. The test kit was used to test the liver glycogen content of mice, and the results were as follows: Fig. 9 Compared with the mice in the control group, the liver glycogen storage content of the mice in the hypobaric and hypoxic group after exhaustive running in the high altitude hypoxic environment was significantly increased* (P=0.0230, N≥6). Compared with the mice in the hypobaric and hypoxic group, the liver glycogen storage content of the mice in the selenium treatment group after exhaustive running in the high altitude hypoxic environment was significantly increased** (P=0.0023, N≥6).

[0060] Determination of muscle glycogen content. The test kit was used to test the muscle glycogen content of mice. The results were as follows: Fig.10 Compared with the mice in the control group, the muscle glycogen storage content of the mice in the selenium treatment group after exhaustive running in the high altitude hypoxia environment was significantly increased* (P=0433, N≥6). Compared with the mice in the low pressure and hypoxia group, the muscle glycogen storage content of the mice in the selenium treatment group after exhaustive running in the high altitude hypoxia environment was significantly increased* (P=0.0296, N≥6).

[0061] The content of the present invention is not limited to the embodiments listed, and any equivalent changes made to the technical solution of the present invention by ordinary technicians in this field after reading the description of the present invention are covered by the claims of the present invention.

Claims

1. A method for preparing a selenium-enriched Lactobacillus rhamnosus microecological preparation, characterized in that: The following steps are involved: Step 1: inoculating Lactobacillus rhamnosus SHA113 into a liquid culture medium to obtain a bacterial suspension; Step 2: adding sodium selenite to the bacterial suspension in step 1 to obtain selenium-enriched Lactobacillus rhamnosus; Step 3: centrifuge the selenium-enriched Lactobacillus rhamnosus obtained in step 2, discard the supernatant, and collect the precipitate; Step 4: Wash the collected precipitate with sterile water; Step 5: crushing the washed precipitate; Step 6: Dry the crushed precipitate into powder to obtain a selenium-rich microecological preparation.

2. The method for preparing the selenium-enriched Lactobacillus rhamnosus microecological preparation according to claim 1, characterized in that: The Lactobacillus rhamnosus SHA113 in the step 1 was deposited in the China Center for Type Microorganism Collection on December 25, 2017, with a deposit number of CCTCC NO: M 2017838.

3. The method for preparing the selenium-enriched Lactobacillus rhamnosus microecological preparation according to claim 2, characterized in that: In the step 1, the inoculation amount of Lactobacillus rhamnosus is 1% v / v to 10% v / v, and the culture conditions are static culture at 37° C. or culture at 50 rpm to 250 rpm for 24 to 48 hours.

4. The method for preparing the selenium-enriched Lactobacillus rhamnosus microecological preparation according to claim 3, characterized in that: In the step 2, the concentration of sodium selenite is 1.0 mM to 10.0 mM.

5. The method for preparing the selenium-enriched Lactobacillus rhamnosus microecological preparation according to claim 4, characterized in that: In the step 3, the centrifugal speed is 4000 rpm / min to 10000 rpm / min, and the centrifugal time is 3 min to 10 min.

6. The method for preparing the selenium-enriched Lactobacillus rhamnosus microecological preparation according to claim 5, characterized in that: In the step 5, the crushing method is ultrasonic crushing or grinding crushing.

7. The method for preparing the selenium-enriched Lactobacillus rhamnosus microecological preparation according to claim 6, characterized in that: The selenium content in the selenium-rich microecological preparation obtained in step six is ​​10 mg / g to 35 mg / g.

8. A selenium-enriched Lactobacillus rhamnosus microecological preparation obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the selenium-enriched Lactobacillus rhamnosus microecological preparation as claimed in claim 8 in the preparation of drugs and health products for alleviating liver damage in a low-pressure and low-oxygen environment.

10. Use of the selenium-enriched Lactobacillus rhamnosus microecological preparation according to claim 8 in the preparation of medicines and health products for alleviating altitude sickness and improving the body's motor ability under high altitude hypoxia.

Images